PP2A is a major Ser/Thr Protein Phosphatase that functions as a trimeric holoenzyme consisting of a scaffold protein (A), which bridges a catalytic subunit (C) and a regulatory B subunit. The regulatory subunit is thought to mediate substrate specificity and integrate regulatory inputs from a variety of signaling pathways. B55? is a ubiquitously expressed regulatory subunit of the B/B55 family and has been reported to target a considerable number of key substrates with critical functions in cell division, differentiation, survival, as well as tissue-specific specialized processes. The structure of B55? complexed with the PP2A-AC core dimer has been solved. B55? is a WD40 ?-propeller with an acidic surface surrounding the central hole and a marked groove on top of blades 3 and 4. While studies with other ?-propeller proteins have identified the region near the central hole as their binding site, the more convoluted surface of B55? suggests a more complex set of substrate/regulatory subunit contacts. We have previously shown that the retinoblastoma related protein p107 is a substrate of PP2A/B55? and that p107 is dephosphorylated in response to FGF signaling in chondrocytes with kinetics comparable to other unrelated key PP2A/B55? substrates in the MAP kinase pathway, RAF1 and KSR1. Additionally, a previous study had identified domains in the microtubule-associated protein TAU rich in positively charged Lys residues that are required for dephosphorylation by B55?/PP2A. This led to speculation that the positively charged residues in the two binding domains of TAU may interact with the negatively charged residues on the top of B55?. With the aim of understanding how B55? binds substrates, we have performed extensive structure/function mutation analysis of both p107 and B55?. Our results show that two separate regions within the intrinsically disordered spacer of p107 (R1 and R2) are involved in contacting B55?, and both domains contain positively charged residues that are important for binding. We have also found that several of the acidic residues in the top of B55? reported to be critical for TAU dephosphorylation are dispensable for p107 binding. Our preliminary data suggest limited overlap in the surfaces contacting TAU and p107. While the crystal structure of the PP2A/B55 holoenzyme has been instrumental in developing a hypothetical mechanism for TAU recognition, it is not known how the surface of B55? can bind such a variety of unrelated substrates, and the motifs that characterize these substrates remain elusive. Our hypotheses is that B55?/PP2A substrate specificity is mediated by discriminator surfaces within the top groove and hole of B55? that consist of substrate specific combinations of residues targeting clusters of positively charged residues within intrinsically disordered domains in substrates. This mechanism allows the B55? surface to orient substrates such that the phosphosite is positioned in the enzyme active site. Therefore, the overall goal of this proposal is to determine how the surface of B55? can bind unrelated substrates and to develop a detailed model consistent with experimental data (both existing and to be obtained under this proposal), with an initial focus on p107 and subsequently extended to other substrates. This will be accomplished with the following aims: (1). To identify the determinants of substrate specificity in B55? that mediate binding to substrate specific motifs in p107 and build a testable high-resolution docking model. (2). To identify the specific binding surfaces on the B55? ?-propeller top that discriminate unrelated substrates (e.g., RAF1, KSR1, TAU and a model mitotic substrate, PRC1) to build a high-resolution model that explains presentation of the phosphorylated residue to the PP2A/C catalytic site. (3). To identify the extent of substrate diversity in select cell types using solution based proteomics analysis and to establish how perturbation on B55? affects substrate binding and overall cellular signaling. Understanding the determinants of substrate specificity for this PP2A holoenzyme is a fundamental question in eukaryotic signaling with important therapeutic implications as modulation of PP2A activity is actively being explored in a variety of cancers. Moreover, PP2A/B55? has been found to be down regulated in Alzheimer disease, where TAU is found abnormally hyperphosphorylated and aggregates.